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Laboratory Diagnosis of Human Immuno Deficiencv Virus Infection

Donald S. BurkeF ,'PE OF 7EOR7. 3b .,VE COVERED -F4 DATE OF DEPCRT Ycir V.Cn,!-ai zE ~~'anuscript -RO% T___ O ____ 1989, September -

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E_0 GROUP SijR-GR~oup _Diagnosis of HIV, -AIDS Diagnosisr-e,.-Laboratory Diagnosis of HIV-

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Laboratory Diagnosis of HumanImmunodeficiency Virus Infection

Donald S. Burke, MD*

The acquired immune deficiency syndrome (AIDS) is the clinicallyapparent preterminal stage of a prolonged infection with the humanimmunodeficiency virus (HIV), most often manifested as Pneumocystiscarinii pneumonia, Kaposi's sarcoma, or other opportunistic infections ormalignancies. AIDS is by definition a clinical syndrome diagnosis.

This article deals exclusively with the diagnosis of HIV infection, notwith AIDS, and reflects the author's belief that quality medical caredemands a precise etiologic diagnosis. A United States PresidentialCommission recently stated that "The term AIDS is obsolete." 9 8 No-where is this more true than in the diagnostic laboratory.

THE VIRUS AND CLINICAL COURSE

HIV type 1 (HIV-1), a retrovirus of the Family Lentiviridae, is anenveloped, RNA-containing virus with a diameter of 80 to 120 nm.44

Two glycoproteins are exposed on the viral membrane, gpl20 andgp41.s The gpl 20 (external membrane glycoprotein) includes the aminoacid sequences responsible for virion binding to the CD4 molecule onthe surface of T-helper cells. The gp4l (transmembrane protein) isthought to anchor gp 120 to the virion surface and to be involved in fusionof the viral and target cell membranes. Within the membrane is a nucleo-capsid in the shape of a truncated cone.

Two nonglycosylated proteins, p24 and p 17, are important internalstructural proteins.3 6 Reverse transcriptase, the enzyme protein thatsynthesizes DNA from the virion RNA template, is carried in the virioncore along with the RNA.

3 7

The genome of HIV contains at least nine distinct genes that encodepolypeptides. Three of these, the gag, pol, and env genes, code for thestructural proteins.48 The gag (group antigen) gene codes for the core

* Director, Division of Retrovirology, Walter Reed Army Institute of Research, WalterReed Army Medical Center, Washington, DC

All material in this article is in the public domain with the exception of any borrowed tables

and figures.

Clinics in Laboratory Medicine-Vol. 9, No. 3, September 1989 369

370 DoN&W S. Buaix

proteins p17 and p24, as well as their common precursor p55; the pol(polymerase) gene codes for proteins with proteinase, reverse transcrip-tase, and endonuclease activity; and the env gene codes for the externalmembrane (gpl 20) and transmembrane (gp41) proteins. In addition tothe structural genes, there are at least six other genomic open readingframes that encode proteins which are thought to be involved in regula-tion of virus growth through activation or suppression of transcription ortranslation of the viral structural genes (Table 1).

Although HIV was initially thought to be primarily an infection ofCD4 positive T-helper lymphocytes, it is now clear that cells of themonocyte/macrophage lineage can support virus growth in vitro and invivo. 4'64 9 Some continuous cell lines derived from other organs, such asintestinal epithelial cells, can also be permissive for HIV. Cell activationand proliferation accelerate virus replication.

In the hours and days immediately following HIV infection, the virusprobably first gains entry into and replicates in mononuclear cells in themucous membranes lining the genital tract and in mononuclear cells andfollicular dendritic cells of local lymph nodes."6 Within one to a fewweeks HIV appears free in the plasma and within circulating mononu-clear cells. There is no detectable antibody response during this so-calledseronegative viremic phase or "window." 5 7 HIV antibodies typicallyfirst appear in the blood 4 to 12 weeks after initial infection, but rarelythe "window" and may last up to 6 months or longer. As antibodies to theenv and gag and then pol gene products arise, viral antigens decline anddisappear; virus isolation from the blood becomes more difficult.30 Theacute phase of infection is usually asymptomatic, but 20 per cent ofpatients may experience a nondescript illness characterized by low-grade fever, fine maculopapular rash, and diarrhea.5 .94 Severe illnessduring the acute phase of infection, manifested by fever and encephalop-athy, is relatively rare. 5

Table 1. The Known Genes of HIV-1 and the Proteins Encoded by These Genes

GENE NAME FUNCTION

Structural Genes (Products are released with newly formed virions)GAG Core proteins (p55, p2 4, p17)POL Viral enzymes (p64, p53, p31)ENV En, 'c - )roteins (gpl60, gpl20, gp4l)

Regulatory Genes (Products art onb - d to host cell)TAT Trai,. . , orREV. Reguia. f virion protein expressionVIF f Virion infectivity factorNEFt Negative regulatory factorVPR ? UnknownVPU ? Unknown

Note that only the structural proteins are present in purified virion preparations.Purified virions are used in preparation of conventional Western blot strips; as a conse-quence, only antibodies to the virion structural proteins can be detected in conventionalHIV- 1 Western blot strips.

* Formerly named ART or TRS.Formerly named SOR.

I Formerly named 3'-ORF.

LABORATORY DIAGNOSIS OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION 371

A prolonged phase follows in which the patient is seropositive butasymptomatic. Despite the absence of symptoms, the chronic HIV infec-tion induces subtle and progressive immune dysfunction, which may beclinically manifest only as lymphadenopathy or which may be clinicallytotally silent.5 3.82 Viral antigens can be demonstrated primarily in follicu-lar dendritic cells. Over the next several years numerous immune defectsappear, the most prominent being the progressive loss of CD4 positiveT-helper lymphocytes. When the CD4 T-helper lymphocyte countdrops below 100 per cubic mm, overt manifestations of HIV infection areimminent, typically in the form of opportunistic infections. Severe im-munocompromise is accompanied by a decline in HIV antibodies, espe-cially a pronounced loss of anti-gag, and a rise in HIV antigen. 54.82 Duringthe late stage of infection, virus can again be easily isolated from blood.

ANTIBODY TESTS

SCREENING ASSAYS

Screening assays for detection of HIV antibodies have in commonthe properties of high sensitivity, ease of performance, rapidity, and lowcost. In the United States the only assays presently available for screen-ing are enzyme-linked immunosorbent assays (ELISAs), which use ly-sates of partially purified whole virions as a source of viral antigens forsensitization of a solid plastic surface (such as a microtiter plate well or abead). HIV-specific human antibodies in the test serum specimen arebound to viral antigens on the plastic surface and detected with com-plexes of enzymes and animal antibodies that specifically recognizehuman immunoglobulins. Enzymatic activity bound to the surface is inturn determined with a soluble substrate that is cleaved and changescolor. The resultant color change (change in optical density or OD) isdirectly proportional to the concentration of HIV antibodies in the testserum specimen. Whole virus lysate ELISAs were first licensed in early1985 for the purpose of screening donated blood. These assays promptlyfound wide application as the initial step in algorithms designed for thediagnosis of HIV infections.

An HIV whole virus lysate ELISA should not be used as a "standalone" test for diagnosing HIV. Although the specificity of this type ofassay is quite good (on the order of 99.5 per cent for most commercialkits), some true negative samples do give reproducible falsely reactiveresults. 455 6 .84 In most cases the cause of the false reactivity cannot bereadily discerned, but associations with autoimmune diseases and withmultiparity (among women) have been reported .' 18 Depending on theprevalence of HIV in the population being screened, the predictive valueof a repeatedly reactive ELISA may be greater than 90 per cent (forexample, in a clinic for homosexual men in New York) or lower than 10per cent (for example, in a blood bank in North Dakota). False reactivityis thought to be caused by antibody binding to cellular proteins thatcontaminate the purified virions. Some of the contaminating cellularproteins may actually be cell surface proteins that become incorporated

372 DONALD S. BURKE

into the virion membrane during the process of viral budding from cellmembranes.

More recently, screening assays based on agglutination of small par-ticles (red blood cells or latex particles) have been developed.1 0 2 Incontrast to ELISAs, which require equipment for measurement of opticaldensities, agglutination reactions can be scored by the naked eye, adistindt advantage in a field setting. However, agglutination reactionscan be difficult to interpret and require trained observers. Althoughprecise data are not yet available, it seems unlikely that a subjectivelyscored agglutination test will ever achieve performance equal to thequantitatively scored ELISA. The same constraints that apply to thepredictive value of a positive ELISA, will also limit the utility of aggluti-nation reactions for diagnosing HIV. In most circumstances suboptimalspecificity cannt be tolerated; therefore, a positive agglutination testshould also be confirmed with an independent, and preferably morespecific, assay method.

CONFIRMATORY TESTS

Despite the preference of some for the term supplemental test, thisauthor is of the opinion that "supplemental" connotes a level of uncer-tainty which is not conveyed in the term "confirmatory." Since theseassays are widely used to establish a firm diagnosis of HIV in the clinicalpractice of medicine, tbe term confirmatory seems to be more appropri-ate and, thus, is used in this article.

Western Blot

The Western blot, or immunoblot, is the most widely used confirma-tory test for the detection of HIV antibodies.95 As in the screeningELISA, the source of HIV antigens is partially purified whole virionsharvested from in vitro cultures of infected human lymphocyte cell lines.Lysed virus is "vertically" electrophoresed through a sizing polyacryl-amide gel, and the separated and banded viral proteins are "horizon-tally" electroblotted from within the gel onto the surface of nitrocellu-lose paper. The paper is cut into several strips, each of which hasessentially identically located bands of viral proteins. (Although viralband positions on paper strips derived from a single sizing gel run areidentically located, slight alterations in electrophoretic conditions fromrun to run may result in minor alterations in the migration distances ofviral proteins from run to run. For this reason, interpretation of bandingpatterns is facilitated by testing of positive and negative control speci-mens using strips from the same run that are used for the unknownsamples.) Note that contaminating cellular proteins in the virion prepa-ration are also electrophoresed, transblotted, and distributed on thepaper strips with the viral proteins. One paper strip is then used as thesolid phase to detect antiviral antibodies using reagents and methodssimilar to the ELISA: binding of HIV-specific antibodies present in thetest serum specimen to proteins on the Western blot strip eventuates in acolor change at those locations (band) where the antibodies have bound.

LABORATORY DIAGNOSIS OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION 373

Most Western blot strips prepared in the United States before 1987lacked appreciable bands corresponding to the high molecular weightviral envelope proteins (gp 120 and its precursor gp 160).2 4 Underrepre-sentation of these proteins was probably owing to several factors, espe-cially loss of gpl 20 from the virion surface due to shearing during purifi-cation by ultracentrifugation, and by denaturing of antigenic activityduring electrophoresis and transblotting. Since early 1987 blot prepara-tion methods have been modified to ensure that the high molecularweight envelope bands can be clearly identified with most patient sera.

Strongly positive HIV Western blots show dark bands at positionscorresponding to the all the major gag (p55, p24, and p17), pol (p64,p53, and p31) and env (gpl60, gpl20, and gp4l) bands. Gag and polbands are relatively narrow with sharp edges, whereas the glycosylatedenv bands tend to be broader with indistinct upper and lower margins(the gpl60 and gpl20 bands often overlap). Presence of the gag (p55)and env (gpl60) precursor proteins on Western blots supposedly de-rived from purified virions suggests incomplete purification of virionfrom intracelluiar proteins, but the possibility that some structural pro-teins can be incorporated into intact virions without uniform cleavagehas not been ruled out. Nonstructural regulatory HIV encoded proteinsare separated from the virions during purification, and antibodies tothese proteins are not identifiable on standard Western blots.

Western blots performed using antibody negative sera usually showno bands and are uniformly "snowy white" for their entire length. Allnine viral structural proteins are present on the strip surface, but thebands remain colorless because no antibodies have bound. With somestrongly reactive sera all nine bands are darkly stained. However, not allsera from definitely infected persons show reactivity with all nine HIVstructural proteins on Western blot, and not all sera from definitelyuninfected persons produce blank strips. For this reason, interpretivecriteria must be used to evaluate the significance of various Western blotpatterns.

Before 1987 (and before antibodies to gpl60 and gpl20 could beregularly visualized by blot), criteria informally presented by the U.S.Public Health Service were widely used (p24 or gp41, CDC; p 2 4 andgp4l, NIH) to define a positive Western blot. In U.S. Department ofDefense HIV testing programs, somewhat more specific criteria wereemaployed (p24 + p55 and/or gp4 1). However, it quickly became appar-ent that all of these criteria sets were less than perfectly specific. Falsepositive gag p24 reactivity was a major problem, and some cases withfalsely reactive bands at both p24 and p55 were identified.

Regrettably, there are still no U.S. nationally (or internationally)agreed upon criteria for Western blot interpretation. Several groupsconcerned with HIV serodiagnosis currently use different sets of inter-pretive criteria. All agree that a snow white, clean lane pattern is nega-tive. However, there are major differences in the definition of positiveblot (Table 2). Some criteria currently in use include:

1. The Association of State and Territorial Public Health Laboratory Direc-tors and the Department of Defense: two or three of three bands among p2 4,gp41, gpl20/160

374 DONALD S. BuRKE

Table 2. Six Different Sets of Criteria for a Positive Western Blot

YEAR AGENCY CRITERIA FOR POSMVE WESTERN BLOT

&e Cpl2O-Gpl6O1984 CDC 24 or 411985 NIH 24 and 411985 DOD (24 and 55) or 41Post Gpl20-Gpl601987 ASTPHLD 24

41 2 of 3120-160

1987 FDA 2431 3 of 341-120-160

1987 Red Cross 15-24-5531-53-64 3 of 341-120-160

2. The American Red Cross: at least one gag (p55, p24, p1 7 ), one pol (p6 4,p53, p31), and one env (gp41, gp120/160) band

3. The Food and Drug Administration (FDA; per the package insert in thelicensed DuPont Western blot kit): three of three among p2 4 , p31, (gp4l and/orgp120/160)

The estimated seitsitivity and specificity of these Western blot in-terpretive criteria are presented in Figure 1. The old and now aban-doned CDC criteria were highly sensitive but poorly specific. Con-versely, current FDA critena (on the package insert) are quite specificbut show poor sensitivity.

By convention, all strips that do not meet the criteria for a positiveWestern blot but that do show one or more bands at molecular weightscorresponding to HIV proteins are termed reactive but nondiagnostic orindcterminant. Up to 15 per cent of sera from normal noninfected per-sons when tested by the FDA licensed commercial Western blot (Du-Pont) show a reactive but nondiagnostic (indeterminant) pattern. Inde-terminant Western blot patterns are found as commonly amongspecimens that are negative by screening ELISA as among specimensthat are screening ELISA-reactive. Most indeterminant blots show only aweak gag band, most commonly at p17 or at p24. 24,7 7 '9 7 Other singleband, and occasionally multiple band, indeterminant blots are less fre-quent. When a new blood sample is drawn and immediately tested from aperson with a recent indeterminant blot result, the repeat is usuallycompletely negative. However, many normal persons do have repeat-edly positive, "gag only" indeterminant Western blots. 22 The signifi-cance of an indeterminant blot is at present poorly understood. Many,especially those with weak and transient reactivity, are probably causedby technical errors of specimen contamination by pipette tip carryoveror splashing. Rarely, an indeterminant blot signifies a specimen obtainedearly during seroconversion to HIV. Other intriguing possibilities exist,such as prior infection with an as-yet unidentified related retrovirus, orautoantibodies to antigenically related epitopes on normal human pro-teins, but these currently remain in the realm of theoretic possibilities.

LABORATORY DIAGNOSIS OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION 375

SPECIFICITY

100FDA

rN IH_

98 AST

r AR C1

96

DOD

94

92

90

88 ICDC90 92 94 96 98 100

SENSITIVITYFigure 1. Sensitivity and specificity of various criteria for interpreting HIV-I West-

ern blot. True status of specimens determined by repeated Western blot testing, results oftesting by antibody assays constructed from molecularly cloned and expressed envelopepolypeptides, and results of radioimmunoprecipitation assays. For exact definitions of eachof the six interpretive criteria shown see Table 2.

376 DoNALD S. BosKE:

Approael.es to further testing and reporting of indeterminant West-ern blot results are provided in detail in the discussion of screening in lowprevalence populations.Immunoassays Constructed from Antigens Produced Through

Recombinant DNA Technology or by Chemical Synthesis

In the few short years since the first isolation of HTLV-IIIB and LAV,the complete or partial nucleotide sequence of the genomes of at least 17HIV strains has been determined. The genes for the structural proteinshave been located, and hypervariable, variable, and highly conservednucleotide sequences have been identified within these genes.' 7 Thisdetailed genetic information has been used to construct a variety ofELISAs for detection of HIV antibodies in which molecularly cloned andexpressed antigens or synthetic antigens are used in place of the wholevirus lysate antigens.

The first molecularly defined antigens used in construction of diag-nostic ELISAs were selected largely by guesswork. Since it was knownthat antibodies to the env gene proteins appear early during seroconver-sion and persist until death, various constructs which included conservedregions of the env gene were cloned and expressed in Escherichia coli.This was done without knowledge of exactly which regions of the envgene were immunodominant. Conserved sequences (nucleotide se-quences found essentially identical in all known HIV strains) were em-phasized to ensure that all human sera would be reactive with the ex-pressed antigen. One such ELISA produced from a molecularly clonedand expressed antigen spanning the carboxy!-terminal third of gp120and the amino-terminal half of gp41 (CBre3, produced by CambridgeBiosciences, Inc.) was exhaustively evaluated and found to be compara-ble or superior to the Western blot in its sensitivity and specificity.2"Because the gene was expressed in E. coli, the problem of false reactivitywith lymphocyte proteins was eliminated. (The analogous problem offalse reactivity with contaminating E. coli proteins was minimized byantigen purification; removal of E. coli proteins from the antigen wasessentially complete, as proven by absence of reactivity of sera frompatients convalescent from E. coii sepsis.) The CBre3 env ELISA hasbeen used extensively as a second confirmatory test in Department ofDefense HIV testing programs with excellent results. Other assay s _on-structed from molecularly cloned and expressed HIV antigens have beenproduced and are currently undergoing clinical trials. Various strategieshave been used to construct assays: only a single antigen (env) in anELISA format, a single antigen (env) in a dot-blot or slot-blot format, twofused antigens (env and gag) in an ELISA format, multiple antigens (gag,pol, and env) in separate wells in an ELISA format, or multiple antigens(gag, pol, and env) in a multi-slot-blot "pseudo-Western blot" format.

To date none of these assays, including the CBre3 env assay, hasbeen licensed by the FDA. However, because the performance of thistype of assay appears to be excellent, safety concerns during antigenproduction are nil, and costs of antigen preparation can be lowered, itseems likely that molecularly cloned and expressed antigens will rapidlyreplace virion derived antigens in the construction of diagnostic assays

I 'A)R.TORY Dik(:%osts OF HUMAN IMMLNODEFICIEN(Y Vl', INFE('TION 377

for lilY antibodies. Whether in the future assays constructed from mo-lecularly cloned and expressed antigens will be used predominantly ascoiifirmatorv assavs or as screening assavs will be determined largely bycost[ con-*

: ratioills.

' ,ntrast to molecularly cloned and expressed antigens, which aretyt,, i.d several hundred amino acids long and contain several epitopes.synthetic peptides are, usually only 15 to 30 amino acids in length andencompass only one or two epitopes. Intensive efforts to pinpoint im-munodominaiit epitopes (both for purposes of vaccine development aswell as for diagnostic assay development) have resulted in the identifica-tion of several synthetic peptides that may serve as antigen surro-

gates. 29,5 1.89,90

One epitope, located at the position corresponding to amino acidsnumlers 5S2 to 600 on env gene, is clearly the most immunodominantepitope identified to (late. Essentially all seropositive persons in NorthAmerica have antibodies that recognize and bind to this peptide. Indeed.on the basis of available data, it appears that all molecularly cloned andexpressed antigens that include this epitope are excellent for use indiagnostic assay construction, whereas those that do not include thisepitope have suboptimal sensitivity. Minor differences in nucleotidesequence between HIV strains from Africa and those in the United Statesrender this pepti(le less than perfectly sensitive for detecting seruposi-tive Africans.5 0 It would seem imp, udent to base a diagnostic test for HIVon a single epitope, should the strains prevalent in the United Statesmutate at this site, a disastrous loss in test sensitivity could ensue. How-ever, a diagnostic assay constructed from a "cocktail" or two or moresynthetic peptides is a realistic alternative. More likely is the use of singleepitope synthetic peptide antigens in the construction of assays for de-termining stage and progrosi.

Indirect Immunofluorescent Antibody Test (IFA)

Some laboratories with extensive IFA experience have found thistechnique useful for confirming the presence of HIV antibodies. 67.72,96

Mixtnies of approximately equal numbers of HIk-infected and unin-fected cells are spotted onto slides, reacted with dilutions of the testserum specimen, and stained. Negative samples produce no fluores-cence. False positive reactions are differentiated from true positive reac-tions by cunting the percentage of cells showing fluorescence: 100 percent with falsely reactive specimens and 50 per cent with truly reactivespecimens. When appropriately cont'olled, the assay is reported to showsensitivity and specificity at least compairable to the Western blot.

Radioimmunoprecipitation Antibody (RIPA) Test

Some research-oriented laboratories use the radioimmunoprecipi-tation antibody test to confirm the presence of HIV antibodies. 12.2 .9 3 Inthis technique, radiolabeled amino acids (such as S-35 cysteine) areadded to infected cell cultures. The labeled amino acids are incorporatedinto polypeptides that are being actively translated. By selection of thecorrect labeled amino acid and correct time interval from label addition,intrinsically labeled viral proteins with high specific activity can be oi-

378 DONALD S. BURKE

tained. Patient sera are added to the clarified labeled cell lysate, andantigen-antibody complexes are precipitated from solution. The result-ant precipitate is electrophoresed over a polyacrylamide sizing gel, andbands are detected by autoradiography. Interpretive criteria for RIPAsvary from laboratory to laboratory, depending on which viral proteinsare preferentially labeled. In the author's laboratory conditions wereselected to optimally label the large envelope glycoproteins gp160 andgp120; the presence of bands at both gp160 and gp120 is necessary andsufficient for a positive diagnosis. The sensitivity and specificity of thetechnique have not been formally evaluated. There is some evidence thatabsence of antibodies to the carboxyl-terminal portion of gp 120, as de-

tected by RIPA, is associated with disease progression.

Other Diagnostic HIV Antibody Tests

In addition to the screening ELISA, Western blot, assays based onmolecularly cloned and expressed or synthetic antigens, IFA, and RIPA,a number of other assays have been used to detect HIV antibodies. Mostnotab'e among these are the IgM capture assay and assays to detect HIVantibody synthesis in vitro.

Efforts to develop IgM capture assays for HIV antibodies have metwith only marginal success.'15 8 , 43 ,6 6 ,74 ,7 6 ,8 1 In contrast to other chronicviral illnesses such as hepatitis B, the signal to noise (P/N) ratio is typi-cally quite low, even during seroconversion; indeed many groups havefailed to detect specific antibodies with this technique. Perhaps IgMassays using defined synthetic epitopes may show improved sensitivity.

The problem of differentiating between passively acquired vcrsusactively produced antibodies in neonates born to HIV-infected mothershas led to the development of a technique for detecting HIV antibodysynthesis in vitro in cultures of peripheral blood leukocytes from theneonate., 3 5 The technique, involving culture of patient cells, is cumber-some but shows promise.

"FuNCTIONAL" ANTIBODY TESTS

In contrast to the assays noted earlier, which simply detect antibod-ies that bind to HIV antigens, several methods have been reported formeasuring antibody activities thought to have in vivo significance.Among these are virus neutralization, reverse transcriptase inhibition,and antibody-dependent cell-mediated cytotoxicity. Although one orseveral of these assays may eventually be proven mechanistically rele-vant to protection against infection or protection against disease progres-sion, tbere is at present no compelling evidence of such a mechanisticrelationship.

VIRUS ISOLATION

Because isolation of HIV from blood or other clinical specimens istime consuming, expensive, and potentially dangerous, virus isolationcannot be considered a standard diagnostic test for HIV. However. virus

LABORATORY DIAGNOSIS OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION 379

isolation can yield useful information in unusual cases and in clinicalstudies. 0 32 ,3 1.55 .92 All cultures must be handled wit'a strict adherence toproper biocontainment protocols.

The conventional technique is to cocultivate patient peripheralblood mononuclear cells (PBMCs), with interleukin 2 (IL-2) and phyto-hemagglutinin-stimulated normal donor PBMCs, conditions that selec-tively stimulate activation and proliferation of CD4-positive lympho-cytes. Cell culture supernatants are periodically monitored forappearance of HIV antigens and reverse transcriptase (RT) activity. Theminimum number of patient PBMCs required for maximal isolation rateshas not been rigorously established, but as few as 3 X 10s PBMCs (thenumber typically found in 0.3 ml of blood) is usually sufficient; culture ofgreater number of cells does not appreciably improve the probability ofan isolate. Viral antigens or RT is usually detected after 7 to 14 days,rarely after 21 days in culture. Some isolates grow rapidly to high titer,while others grow more slowly. These in vitro differences of growthproperties between strains may be related to differences in the in vivorate of disease progression, but further data are sorely needed on thispoint.

Depletion of CD8-positive cells from the patient PBMC populationis said to improve isolation rates, but this technique has not been widelyvalidated and should still be considered as experimental. Some laborato-ries have used continuous lines of CD4-positive cells as targets, but therehas been no clear demonstration of an advantage of such cell lines overheterologous normal donor PBMCs as targets. HTLV-I transformed cellsor cells stably transfected with the tat-3 gene also show good sensitivityas targets. The concept that some HIV strains have inherent defects inregulatory protein production or activity warrants vigorous investiga-tion.

HIV isolation rates are directly proportional to the stage of illness.23

HIV can be recovered with relative ease fro-,l patients with late stagedisease (essentially 100 per cent of patients), but isolation rates aretypically somewhat lower among early, asymptomatic patients (20 to 60per cent). Although not fully studied, it appears that recovery rates aregood during the antibody negative "window," then decline as antibodiesto the structural proteins rise.3 HIV can often be recovered from plasma,but titers of free virus in plasma are low and isolates are obtained lessoften than with cocultivation of PBMCs. HIV can also be recovered fromcerebrospinal fluid (CSF) in a substantial proportion of patients, eventhose without overt central nervous system manifestations. 7.27 59 Theclinical significance of isolates from the CSF of apparently healthy indi-viduals is at present unknown. The clinical and epidemiologic signifi-cance of scattered reports of inconsistent isolation of HIV from saliva,semen, breast milk, and other body fluids is likewise uncertain.

Although most laboratories use activated CD4-positive lympho-cytes as targets to culture HIV, the monocyte/macrophage is an alterna-Iiv,- target cell for in vitro cultures. Stimulation of PBMCs with macro-phage-colony stimulating factor (M-CSF) may improve isolation ratesand select for strains with relative tropism for macrophages rather than Tcells.

46 .4 9

Virus isolation should be used cautiously as a diagnostic technique.

380 DONALD S. BURKE

Falsely negative cultures are not infrequent, and patients tend to inter-pret a negative culture result as evidence of freedom from infection.False positive culture results are also a distinct possibility in a busyisolation laboratory. Ideally every HIV isolation laboratory should regu-larly receive and blindly process known positive and negative bloodsamples in order to ensure quality results.

DETECTION OF VIRAL ANTIGENS

Conventional "sandwich" antigen detection ELISAs have notproved to be very valuable for detection of HIV antigens in serum orplasma. Limited usefulness can be related to the relatively low signal tonoise ratios obtained with positive samples (in the range of ratios of 2 Xto 5 X). Specimens from patients with late, clinically overt disease areusually positive, and specimens from normal uninfected persons are usu-ally negative. 4,11'6 4,72 ' 99 However, sera from early stage asymptomaticHIV-infected persons are also usually negative. Thus, antigen detectionis not often useful to distinguish true from false reactives among personswith repeatably reactive ELISAs and reactive but nondiagnostic blots.Commercially available HIV antigen kits detect predominantly gag anti-gens. The antigen peak during the "seronegative window" is probablytransient and is detected only infrequently, even among specimens ob-tained from seroconverting individuals in prospectively studied cohorts.The antigen rise in late stage disease is temporally associated with adecline in anti-gag antibodies. However, cause and effect relationshipsbetween rising antigen and declining antibodies in late stage disease areuncertain; it is not known if the antigen rise is due to failure of HIV-spe-cific antibody production or if the decline in anti-gag antibodies is due toaccelerated production of antigens and complexing of HIV antibodiesout of the plasma.6

4

Disappointingly, routine testing of donated blood for HIV antigenshas been unrewarding. Unequivocal positives are exceedingly rare; theyield does not offset the substantial costs.

DETECTION OF VIRAL NUCLEIC ACIDS

In situ hybridization studies, in which radiolabeled cloned HIVDNA is used to probe PBMCs, have demonstrated that cells are rarely (1in 104 to 1 0') labeled. This observation suggests that only a small fractionof PBMCs are actively infected (transcribing HIV RNA) at any giventime.8 5 However, this technique is not sufficiently sensitive to detectHIV proviral DNA sequences, which may be stably integrated into hostcell genomes at low copy numbers such as one or two copies per cell.Measurements of the number of cells showing positive signals by in situhybridization should not be used to draw conclusions about the propor-tion of cells that are latently infected. In any case, in situ hybridization ofblood cells is not a promising technique for diagnostic purposes.

Gene amplification by the polymerase chain reaction or PCR tech-

LABORATORY DIAGNOSIS OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION 381

nique shows considerable promise.87 In the PCR, a pair of synthetic 20 to30 mer oligonucleotide primers, which correspond to highly conservedHIV sequences located 200 to 300 nucleotides apart, are prepared. Inthe presence of the primer pairs, the nucleic acids in the test specimenare subjected to successive rounds of melting and copying. (The processcan be technically simplified by use of a thermostable polymerase.) IfHIV DNA is present in the sample, the sequence intervening betweenthe primer pairs is amplified geometrically. If no HIV DNA is present,little or no DNA is synthesized. Amplified HIV DNA in the reactionmixture can then be detected with a radiolabeled probe that correspondsto the center of the amplified nucleotide sequence. PCR appears to havea sensitivity superior to virus isolation, especially among patients withearly stage illness.'16 2,63,75 ,79 In the author's limited experience, PCR hasshown a sensitivity of 96 per cent among patients known to be infectedand a specificity of 100 per cent among negative controls. PCR is not asimple technique at present, but efforts to automate the procedure areunderway.

STAGE OF ILLNESS AND PROGNOSIS

HIV principally affects the immune system. Any rational system fordisease classification or staging should reflect the extent of end-organdamage. The system used within the Department of Defense is theWalter Reed Staging Classification System, which defines six stages, onethrough six, that reflect progressive stages of immune impairment. Thispathophysiology-based system has been described in detail elsewhere.

8 3

Other HIV disease classification systems, such as the CDC system, arebased entirely on clinically overt manifestations and ignore criticallyimportant information that can be readily generated by the clinical pa-thology laboratory.

Accurate measurement of the number of CD4 lymphocytes percubic millimeter of blood is a key measure of the stage of illness .3 3 .4 7 ,5 2 .A severely depressed CD4 count is the single best predictor of imminentopportunistic infection, and a rise in the CD4 count is associated with thetherapeutic affect of azidothymidine or other antiretroviral drugs. Pa-tient management is markedly improved if the attending physicianknows the CD4 count: immediate hospitalization and aggressive andinvasive diagnostic procedures (such as bronchoscopy and lung biopsy)may be warranted in a patient with a depressed CD4 count, but conven-tional treatments and outpatient observation almost always suffice forthe management of patients with normal CD4 counts.

8 2

Quantitative measurement of the CD4 count is conventionally per-formed by flow cytometry. Peripheral blood leukocytes are stained withfluorescence-tagged monoclonal antibodies, and the percentage of lym-phocytes stained with anti-CD4 is measured by sensors in the flow cy-tometer. The total number of CD4-positive lymphocytes per cubic milli-meter of blood is derived by calculation: Total white bloodcells X Percent of lymphocytes among white cells X Per cent of lymphocytesstained with CD4.

382 DONALD S. BURKE

The CD4 count must be measured carefully. Several factors havebeen identified that contribute to imprecision of the CD4 count. (1) CD4counts vary with a diurnal cycle, with peak values in the evening as muchas double those of nadir values in the morning. (2) Technical variations inpreparation, staining, and fractionating of cells by flow cytometry can besubstantial; however, if appropriately standardized, the coefficient ofvariation (CV) of flow cytometric measurements from laboratory to labo-ratory can be kept below 3 per cent (3) Surprisingly, imprecision inconventional white blood cell count and differential cell counts is a majorsource of variability (CVs on the order of 25 per cent). (4) Acute minorviral infections can transiently lower the CD4 count8 9 ; a depressed countshould be validated by retesting 3 months later.

Other measurements of immune dysfunction can also be associatedwith the stage of illness, such as blood concentrations of immunoglobelinisotypes, blood beta2-microglobulin concentrations, and blood and uri-nary neopterine levels, but these laboratory measurements have notfound wide usage among practitioners.3 9' 42 ' 63 80"10 3 Many cliniciansprefer to use the ratio CD4/CD8 lymphocytes rather than the absolutenumber of CD4 cells per cubic millimeter, but the CD8 count is rela-tively labile and devalues the information expressed by the CD4 countalone.

DIAGNOSIS OF HIV IN SPECIAL SETTINGS

SCREENING IN Low PREVALENCE POPULATIONS

HIV antibody testing with intensive education and counselling ofinfected persons can be an important component of a comprehensivepublic health program for control of the HIV epidemic. In those settingsin which HIV testing is not performed because of specific clinical indica-tions, the diagnosis of HIV infection rests entirely upon the laboratorytest results. As noted earlier, HIV screening whole virus lysate ELISAscannot be used as stand alone tests, owing to the regular occurrence offalsely reactive test results. Depending on the prevalence of HIV in thepopulation being tested, such as blood donors or civilian applicants tomilitary service, the number of persons with falsely reactive screeningELISAs may actually be greater than the number with truly reactiveELISA results. For this reason, every laboratory that performs HIVscreening ELISAs must have an automatic, preset integrated mechanismfor confirmatory testing of ELISA reactive specimens. (The standard,"simplest form" HIV testing algorithm is shown in Figure 2.)

Indeed, most thoughtful directors of HIV screening programs be-lieve that patients should not be informed of a reactive screening ELISAtest result before the specimen has been exhaustively evaluated withconfirmatory tests. By withholding ELISA data until a definitive labora-tory diagnosis is reached, patients can be spared the needless fear andanxiety that is invariably engendered by a "positive AIDS test." Implicitin this approach is a rapid turn-around time for confirmatory testing.

The HIV testing algorithm currently used by the Department of

LABORATORY DIAGNOSIS OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION 383

SCREENING ELISA 1 I

1 0- NON-REACTIVE -- *NEGATIVE

REACTIVE

ISCREENING ELISA 2 AND 31

1 P. NON-REACTIVE --- NEGATIVE

REACTIVE

CONFIRMATORY TEST 1]

------ 1 NON-REACTIVE --- NEGATIVE

REACTIVE

1 0NON-DIAGNOSTIC

POSITIVEFigure 2. HIV Testing Algorithm: Simplest Form. Note that in this "simplest form"

algorithm no method is used to resolve the interpretation of specimens with "nondiagnos-tic" results by confirmatory test, and that a second specimen is not required to verify thediagnosis.

Defense to screen civilian applicants for military service is shown inFigure 3. Several features of this algorithm deserve emphasis:

1. Screening ELISA test results are not referred to as "positive," but asreactive. Nonreactive results are reported out as negative.

2. "Reactive" screening ELISA results are not reported. Test results at thistime are reported as "pending." The Western blot is the standard first confirma-tory test. A clean lane, nonreactive blot is reported out as negative, and a reactiveblot that meets the criteria of the ASTPHLD/DoD (see previous discussion) isreported out as positive.

3. Western blots that produce a reactive but nondiagnostic pattern are sub-jected to a second confirmatory test, the CBre3 molecularly cloned and ex-pressed HIV env ELISA. Most blot reactive but nondiagnostic specimens areeither clearly positive (OD > 1.0) or clearly negative (OD < 0.3) with this test.

384 DONALD S. BURKE

SCREENING ELISA 1I

-l NON-REACUVE -- NEGATIVE

REACTIVE

ISCREENING ELISA 2 AND 3

1 0 NON-REACTIVE -- NEGATIVE

REACTIVE

CONFIRMATORY TEST I

1 - NON-REACTIVE -- NEGATIVE

REACTIVE

RE V - NON-DIAGNOSTIC

CONFIRMATORY TEST 21

POSITIVE NEGATIVE

OBTAIN VERIFICATION SAMPLE

TEST VERIFICATION SAMPLEl

POSITIVE = DIAGNOSIS ESTABLISHEDFigure 3. Complete HIV Testing Algorithm. Note that in this comp!Pte testing algo-

rithm a second confirmatory assay is used to resolve the interpretation of specimens with"nondiagnostic" results by the first confirmatory test. For example, in US Army HIVtesting programs, an immunoassay based on a molecular cloned and expressed HIV enve-lope polypeptide is used to resolve specimens with "nondiagnostic" or "indeterminant"results by Western blot. Also note that a new, second blood specimen must be obtained andshown to be positive before a diagnosis can be considered to be confirmed.

t

LABORATORY DIAGNOSIS OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION 385

Rarely a specimen that is indeterminant by blot is also indeterminant by CBre3ELISA. These cases are examined by RIPA. (Note: Some laboratories use the IFAas a second confirmatory test.) Every effort is made to avoid reporting out a resultas "indeterminant."

4. If a positive diagnosis is made on the basis of testing of the first sample,the patient is advised to submit a second, new, "verification" blood sample fortesting. This is done to eliminate false positive diagnoses that result from speci-men handling, specimen labeling, technical, and reporting errors.

5. A diagnosis of HIV is not considered to be established unless a positiveconfirmatory test is recorded on two independent blood specimens from theindividual.

The rate of false positive diagnoses during the first 2 years of themilitary applicant testing program has been retrospectively measured tobe 1 in 135,000 persons tested; the predictive value of a positive diag-nosis of HIV infection in the program was estimated at 99.5 per cent.

2'These results show that with careful attention to test algorithm designand with intense quality control measures, HIV testing in low prevalencepopulations can be extraordinarily specific.

Although the algorithm appears complex, the logistics and cost ofthe system are quite manageable: test results, including Western blotwhere applicable, are uniformly reported back to the examination sta-tion in less than 72 hours, at a cost to the government of less than $3.00per person tested.

3 4

ACUTE INFECTION

Patients who present with an illness and history compatible withacute HIV infection are a difficult challenge to the diagnostic labora-tory.5 8 ,94 At present the most certain method to establish a diagnosis is todemonstrate a seroconversion from negative to positive, but severalweeks may elapse before antibodies appear. Several alternatives can beattempted, such as detection of viral antigens by sandwich ELISA, de-tection of viral nucleic acids by PCR, or viral culture. However, most ofthese techniques are either relatively insensitive or are not yet fullystandardized, and conservative interpretation of results is warranted.

INFECTED BUT SERONEGATIVE PERSONS

Although most persons develop HIV antibodies within a few weeksor months after initial infection, it has been reported that some personsmay remain infected but seronegative for prolonged periods, some for ayear or longer.6 .'7 The exact proportion of HIV-infected persons whoare seronegative is impossible to determine with available diagnostictechniques. Early reports, using incompletely standardized and qualitycontrolled culture techniques, suggested that 6 to 20 per cent of HIV-in-fected persons might be seronegative.7 ' 71 8 More recent reports, usingthe PCR technique, suggest that less than 1 per cent of HIV-infectedadult men remain seronegative for protracted periods. Anecdotal case

386 DONALD S. BURKE

reports suggest that some children infected perinatally may never de-velop HIV antibodies, but again the exact proportions are unknown.

9

INFECTED INFANTS

Infants born to HIV-infected mothers present a special problem inthat all such infants passively acquire HIV antibodies from their mothervia transplacentil transfer. 8 '8° Irrespective of whether the child is in-fected or not, the child is strongly seropositive at birth. Maternal anti-bodies in the infant's circulation decline with a half-life of approximately30 days; the uninfected child may remain seropositive throughout mostof the first year of life. Special techniques must be used to establish adiagnosis. One approach is to measure synthesis of HIV antibodies bycultures of the infant's PBMCs in vitro. Alternatively, IgM antibodies toHIV have been reported detected in infant sera, but this finding has notbeen widely confirmed. Other approaches are to directly detect virus orviral antigen or nucleic acids.3 5 HIV viral cultures, sandwich antigensassays, and PCR all show promise, but none has been thoroughly stan-dardized for use in infants.

HIV-SECIFIC BUT NONETHELESS FALSE POSITIVE ANTIBODY TESTS

As noted previously, infants can be strongly seropositive but none-theless uninfected as a result of acquisition of maternal antibodics. Ln ananalogous manner, adults can become seropositive but uninfected byparenteral inoculation with HIV antibodies contained in blood prod-ucts.40 ,9 1,1 , 101 Before HIV screening tests were available, most lots ofhepatitis B immune globulin (HBIG) prepared from plasma pools se-lected from hepatitis B virus antibody positive donors were also stronglypositive for antibodies to HIV. False positive antibody test results havebeen reported in persons who received HIV antibody positive HBIG.Most lots of immune serum globulin (ISG) produced in the United Statesin the early 1980s are also reactive by ELISA and Western blot, but thetiters in these products are quite low, insufficient to result in seropositiv-ity in the recipient after the ISG is functionally diluted in vivo. No casesof seropositivity have been reported to be attributable to ISG. The frac-tionation procedures used in the manufacture of human globulin prepa-rations completely inactivate HIV; no cases of HIV infection have beentraced to HBIG, ISG, or other human globulin preparations.

RELATED VIRUSES

Weak serologic cross-reactivity between HTLV-I and HIV- 1(HTLV-III) was reported shortly after the discovery of HIV.9 Such cross-reactivity has not been observed with currently available conventionalHIV serologic assays.

Nonetheless, other viruses more closely related to HIV-1 have been

LABORATORY DIAGNOSIS OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION 387

found that do induce antibodies that cross-react in HIV-1 antibodyassays. Sera from West Africans who are infected with the HIV-2 virustypically produve a reactive result when tested by HIV-1 screeningELISA.2 .31.8 1 HIV-1 Western blots on sera from these HIV-2-infectedpersons produce reactive but nondiagnostic patterns. To date only oneperson who is unequivocally infected with HIV-2 has been detected inthe United States. Hundreds of sera from military applicants with reac-tive but nondiagnostic HJV-I blots have been examined for antibodies toHIV-2; none has been positive. These results suggest that HIV-2 andviruses closely related to HIV-2 are exceedingly rare in the United Statestoday.

CONCLUSION

In this article I have dealt exclusively with the technical issues rele-vant to the laboratory diagnosis of HIV. A summary of the performancecharacteristics of the various HIV diagnostic assays is presented in Table3. Laboratory directors must, however, also be keenly aware of themyriad difficult and complex social, political, legal, and economic issuesthat profoundly affect the operations of the HIV diagnostic labora-tory. 14.8 8 Medical and laboratory personnel must take the lead in defin-ing, and then implementing, diagnostic and treatment procedures that

Table 3. Laboratory Assays for Diagnosis of HIV-1 Infection: Comparison ofFDA Licensure Status, Speed of Performance, and Economy

TYPE OF ASSAY LICENSED SPEED' ECONOMYf

IndirectAntibody Detection

Viral lysate ELISA Yes 4 + 4 +Western blot Yes 3 + 3 +Recombinant ELISA No 3+ 4+Immunofluorescence No 3 + 4 +Radioimmunoprecipitation No 2 + 2 +IgM immunoassay No 3+ 3+In vitro synthesis No 2 + 1 +Functional assays No 2 + 1 +

DirectVirus Isolation

T-cell culture No 1 + 1 +Macrophage culture No 1 + I +

Antigen DetectionSandwich ELISA No 3+ 3+

Nucleic Acid DetectionIn situ hybridization No 2 + 2 +Gene amplification (PCR) No 2 + 2 +

Rough scale of approximate time required before assay is complete and results can bereported for that assay, where 4+ = less than 4 hours; 3+ = 4 to 24 hours; 2+ = 24 to 72hours; 1 + = more than 72 hours.

Rough scale of approximate cost per assay in a laboratory routinely performing thatassay, where 4+ = $1 per assay; 3+ = $1 to $10 per assay; 2+ = $10 to $100 per assay;1 + = more than $100 per assay.

388 DONALD S. BURKE

follow well-established traditions.41 Politicians and lawyers can arguetheir social and legal agendas; medical professionals must exert leader-ship in defining and implementing a medical agenda based on excellenceof me(.,,.al care.

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Department of Academic AffairsWalter Reed Army Institute of ResearchWalter Reed Army Medical CenterWa.shington. D.C. 20307-5100